oxadiazole-piperidine motif by a more constrained,
structurally divergent, spiroisoxazoline scaffold
completely abolished the ability of the compounds
to bind to EthR in vitro, whereas they remained
highly effective in boosting ETH activity against

M. tuberculosis (Fig. 1A).

Because these compounds had no antibacterial activity in the absence of ETH but boosted
ETH activity independently of EthR, we hypothesized that they may trigger an alternative bioactivation pathway for ETH.

To identify this pathway, we studied the impact
of SMARt-420, a representative member of the
spiroisoxazoline family, on the transcriptome of
Mycobacterium bovis Bacillus Calmette-Guérin
(BCG) and compared it with the impact of the
bona fide EthR-inhibitor BDM41906 (23).

When the mycobacteria were treated withBDM41906, overexpression of both ethA andethR was observed (Fig. 1B), which is in agree-ment with previous reports showing that EthRrepresses both ethA and its own expression (20).No other major modification of the transcriptomewas observed, suggesting that the inhibitory activ-ity of BDM41906 is restricted to EthR. In contrastto BDM41906, SMARt-420 only weakly inducedthe expression of ethA and ethR. However,SMARt-420 strongly activated the expression ofthe distantly located group of genes bcg_0107c–bcg_0108c, corresponding to rv0076c–rv0077c (Fig.1C) in M. tuberculosis. According to protein ho-mology, Bcg_0108c is predicted to be a memberof the large family of oxidoreductases (http://enzyme.expasy.org/EC/1.-.-.-), which also includesEthA. In silico analyses revealed that bcg_0108cis neighboring the tetR type transcriptional re-gulator gene bcg_0109 ( http://pfam.xfam.org/family/PF00440), indicating analogies betweenthe rv0076c-rv0078 and the ethR-ethA loci(rv3854c-rv3855). The genetic organization ofthe two loci is also similar: rv0077c and rv0078,like ethA and ethR, are divergent open readingframes, both separated by small intergenic re-gions [76 base pairs (bp) and 62 bp for ethR-ethAand rv0077c-rv0078, respectively] (Fig. 1C). Byanalogy with the transcriptional organizationof the ethA-ethR regulon (20), these observa-tions indicate that Rv0078 might regulate theexpression of rv0077c by binding within theintergenic region. This hypothesis was confirmedwith surface plasmon resonance experiments

Fig. 1. SMARt family of molecules reveals alternative ETH bioactivation pathway. (A) Bidimensional representation of the properties of ETH-boosting compounds (BDM). The x axis indicates the shift in the melting
temperature (DTm) of EthR in the presence of BDM compounds, which translates the capacity of the compounds to bind and thermostabilize EthR in vitro
(values are provided in table S1). The y axis indicates the potency [expressed as
the negative logarithm of the median effective concentration (EC50)] of a panel
of compounds to increase ETH antibacterial activity on M. tuberculosis–infected
macrophages. EC50 is the concentration of compound that allows ETH at

0.1 mg/mL (10 times less than the normal MIC) to inhibit 50% of M. tuberculosisgrowth in macrophages. Blue dots and red dots represent compounds of theoxadiazole-piperidine family (first-generation boosters) and of the spiroisoxazo-line family (SMARt), respectively. (B) RNA-seq analysis of genes that aredifferentially expressed in M. bovis BCG exposed for 24 hours to 25 mMBDM41906 (2) or SMARt-420 (3) in comparison with dimethyl sulfoxide–treated bacteria (1). Only genes showing a minimum twofold change (FC) intranscript abundance in at least one condition are shown. RPKM, reads perkilobase per million mapped reads. Whereas BDM41906 specifically inducethe expression of ethA and ethR, SMARt-420 massively induces the expres-sion of bcg_0108c and bcg_0107c. A weak, but statistically significant, inductionof ethA and ethR is also observed. (C) Comparison of the genetic organizationand predicted function of the corresponding M. tuberculosis loci and proteins.Genes bcg_0107c and bcg_108c correspond to rv0076c and rv0077c, respec-tively. Rv0078 is predicted as a transcriptional repressor of the TetR family.